Abstract Monoamine neurotransmitters, such as dopamine and serotonin, modulate fast synaptic transmission in circuits that mediate many complex behaviors including aggression, sleep, attention, and mood. In psychiatry, many therapeutics target monoamine systems at either the receptors or transporters that mediate and regulate monoamine neurotransmission. The vesicular monoamine transporter (VMAT) is responsible for loading all monoamine neurotransmitters into both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), which mediate synaptic and extrasynaptic release, respectively. However, the functional contribution of each type of vesicular release to circuit function and behavior is unknown. Previous studies in Drosophila have demonstrated that the amount and site of amine release can be altered by mutations in the C-terminal trafficking domain of Drosophila VMAT (DVMAT). In a DVMAT null genetic background, the function of several circuits and behaviors are perturbed, but are rescued by transgenic expression of wild-type and trafficking mutant alleles. However, some behaviors are not rescued by trafficking mutants. Mutations that cause DVMAT to preferentially traffic to LDCVs do not rescue function of the oviposition circuit. This suggests that the oviposition circuit is highly sensitive to the delicate balance between synaptic and extrasynaptic release of the neurotransmitter octopamine (OA). I hypothesize that trafficking mutations in the endogenous DVMAT gene locus will confer circuit dysfunction, resembling genetic rescue experiments. To further test this idea, I propose to create a new genetic model of DVMAT trafficking using CRISPR/Cas9 to alter trafficking signals in the endogenous gene. My preliminary data demonstrate that indel mutations that disrupt the DVMAT C-terminus phenocopy previous experiments. This novel genetic model will be useful to study the effects of mutants at endogenous expression levels, facilitates combinations with genetic and molecular tools for circuit analysis, and represents a new platform for genetic screens to find novel regulators of DVMAT function. I propose to study the contributions of the different modes of monoamine release in the Drosophila melanogaster oviposition microcircuit. I hypothesize that alteration of the mode of amine release will result in patterns of muscular contractions and rhythmic activity in target organs that will differ from wild-type animals. These proposed studies will elucidate novel mechanisms of aminergic signaling and new avenues for the research and development of new therapeutics for psychiatric disorders.